PROJECT SUMMARY (ABSTRACT) Humans have exceedingly limited natural limb regenerative abilities. Limb loss due to injury or disease is a major health problem. About two million Americans currently live with the consequences of limb loss, and this number is expected to rise because of increased prevalence of key risk factors such as diabetes and other diseases that affect vasculature. The consequences of amputation are profound for patients and most must rely on prosthetics, which are not perfect. A regenerative medicine approach may one day be feasible if it were understood how total limb replacement can be naturally achieved. To gain this understanding, we are employing an animal model, the axolotl salamander, which can completely regenerate limbs following amputation, even as adults. Axolotl limbs are anatomically similar to human limbs, and their initial development is similar as well. Thus, they offer a blueprint for how a complex, three-dimensional limb can be regrown and functionally integrated into the existing stump following amputation. Key issues that must be resolved if this paradigm is to be translated into the human forum are how axolotls activate and cultivate the progenitors for the new limb. Additionally, the cellular and molecular forces that might antagonize successful regeneration must also be understood as these might normally exist in human patients and thereby prevent regeneration. Future research could elucidate whether the molecular and cellular forces guiding these events are not activated in mammals, or whether they terminate prematurely, or whether they are overtly blocked by other factors. The approach is to first thoroughly understand how limbs do regenerate, and then later use this information to develop hypotheses for future possible therapies. In this proposal, we leverage our recent finding that axolotls can be compromised in their ability to regenerate limbs following repeated amputation. This finding presents a unique opportunity to identify factors that may be limiting in regeneration or may antagonize it. We will examine activation of progenitor cells following successive amputations to determine if these cells are exhausted in regenerative failure. We will also consider the role of macrophages and myofibroblasts in regenerative failure following repeated amputation. We will test if the regenerative limitations we uncovered operate at a local level, within the limb itself and close to the site of amputation, or if they act more systemically, elsewhere in the body. Finally, we will investigate the activities of two genes whose expression becomes dysregulated following repeated amputation, amphiregulin and eyes absent 2, both of which have human correlates. This research will capitalize on the opportunities presented by our new model with the hope that increased understanding of regenerative limitations will be essential for future regenerative medicine approaches in patients.